PROJECT SUMMARY A key event in the progression of osteoarthritis (OA) is the loss of aggrecan from the extracellular matrix of articular cartilage. In addition to aggrecan, hyaluronan (HA) is also coordinately lost. We have found that experimental removal of HA can mimic OA including enhancing the expression of matrix metalloproteinases and the degradation of aggrecan. In gain-of-function experiments, viral overexpression of the enzyme that synthesizes HA, HA synthase-2 (HAS2) reversed these changes primarily by reducing chondrocyte responsiveness to pro-inflammatory mediators such as IL-1?, TNF-? and lipopolysaccharide (LPS) as well as promoting aggrecan retention. However, although HAS2 overexpression (HAS2-OE) shows potential in vitro, it remains difficult if not impossible to test or apply this approach in vivo. While testing the mechanism of HAS2-OE we made a surprising and counterintuitive observation. The small chemical inhibitor of HA biosynthesis 4-methylumbelliferone (4-MU), a coumarin derivative, partially reduced HA biosynthesis as expected but, did not reverse HAS2- OE effects. Moreover, 4-MU by itself, was highly effective (and additive to HAS2-OE) at reducing chondrocyte responsiveness to pro-inflammatory mediators and was effective even with HAS2 knockdown or knockout. 4-MU treatment blocks aggrecan loss in human OA or bovine cartilage explant cultures?a blockage that we have termed, chondroprotection. This proposal will test in Aim 1 whether mice fed 4-MU are resistant to the development of OA in a model wherein OA is initiated via the surgical destabilization of the medial menisci. Recently, others have shown (in other cell types) that HAS2-OE generates a major stress on intracellular UDP-hexosamine pools resulting in upregulation of HIF-1?, followed by changes in cell metabolism and phenotype. 4-MU inhibits HA synthesis by sequestering large amounts of intracellular UDP-GlcUA. We will test the hypothesis that HAS-OE as well as 4-MU both stress and reduce the intracellular UDP-hexose pools in chondrocytes, resulting in similar metabolic feed-back?responses that include activation of transcriptional factors such as HIF-1? that control oxidative phosphorylation/glycolysis pathways. We hypothesize that, as a part of these changes in UDP- hexose biosynthetic coupled pathways, there is reduced sensitivity of chondrocytes to pro-inflammatory mediators thus also providing chondroprotection. The mechanisms involved and changes in chondrocyte metabolomics will be examined in Aim 2 of this proposal. We will manipulate and enhance these glyco- metabolic states in vivo as well as in vitro. The goal of these studies is to determine what state of glucose utilization or metabolism, induced by flux in the UDP-hexose pools, will contribute to the reduction of OA progression.